Protac——一种新的蛋白C活化剂

Stocker等人于1986年从铜头蛇毒(Agkistrodon C.contorix venom,ACCV)中分离出一种新的蛋白C(Protein C,PC)活化剂——Protac。研究表明,与既往采用的凝血酶-血栓调节素(Thrombin-Throbomodulin,T-TM)复合物相比,Protac对PC的激活特异性强,敏感性高,不需辅因子参加,对于人血浆不受蛋白C抑制物的影响,不同时引起血浆中其他蛋白酶类的活性改变,本身不具直接的促凝、抗凝或酰基水解活性。为此,本文就Protac的理化性质及生物活性作一简述。     

江浙蝮蛇毒抗血小板聚集组分的分离纯化及活性分析

蛇毒，特别是蝰蛇和眼镜蛇家族蛇毒含有相当数量作用于血小板的活性成分，它们可诱导或抑制血小板聚集。一些相关成分应用于临床有望作为有效药物治疗出血或血栓性疾病。江浙蝮蛇属蝰蛇类，是我国特有的蛇种。本研究以是否抑制ADP诱导的血小板聚集作为示踪，从江浙蝮蛇毒中分离纯化出一种血小板聚集抑制剂并对其生化特性做进一步探讨。江浙蝮蛇毒经DEAE-Sepharose CL-6B，Sephadex G-75及在FPLC上利用SP-Sepha-rose和Mono Q层析，得到SDS-PAGE条带均一、分子量为65kD的蛋白质组分。该物质无磷脂酶A2、精氨酸酯酶及纤溶酶活性，具有拮抗ADP、胶原诱导的血小板聚集作用。结论：分子量为65kD的蛋白质组分，可抑制ADP、胶原诱导的血小板聚集，且呈明显的剂量一效应关系。     

从Cohn法组份Ⅳ中分离纯化人血浆蛋白C

目的探索建立1种串联离子交换层析和固相化金属螯合亲和层析从Cohn法组份Ⅳ分离纯化蛋白C的方法 ,降低纯化蛋白C的成本,并实现血浆的综合利用。方法 4℃条件下,将Cohn法组份Ⅳ用PBS缓冲液抽提5h,离心(6500g)40min,上清液依次用0.45μm和0.22μm的滤膜过滤后,超滤透析;再通过串联离子交换层析和固相化金属螯合亲和层析,对蛋白C进行纯化。通过SDS-PAGE鉴定蛋白C的纯度;用考马斯亮蓝法通过紫外分光光度计测定总蛋白含量;通过WHO指定的发色底物法和凝固法(一期法)测定蛋白C活性。结果纯化的蛋白C在SDS-PAGE图谱上呈现1条62kD左右的蛋白带;经过离子交换层析,蛋白C的活性回收率为(48.19±9.22)%,纯化倍数为(3.75±0.56)倍;固相化金属螯合亲和层析后,蛋白C活性回收率为(59.61±5.59)%,纯化倍数为(36.79±3.72)倍;蛋白C总活性回收率为(28.77±9.45)%,纯化倍数为(136.64±6.82)倍,比活性为(11.70±0.89)IU/mg。结论串联离子交换层析和固相化金属螯合亲和层析可以从Cohn法组份Ⅳ中获得比活性较高的蛋白C浓缩物。     

一种新的凝血因子Xa抑制剂—蜱抗凝血肽

作者自蜱浸出液经Sephadex G-50凝胶过滤,离子交换层析和反向高效液相层析(RP-HPLC)分离,提炼出一种纯蛋白制品一纯粹蜱抗凝肽(TAP)。对纯化TAP抑制特性的研究表明,TAP对因子Xa活性的抑制作用与其浓度有关,抑制常数Ki=0.588±0.054nM,并且至少预孵育15分钟才能发挥最大抑制作用。因此,TAP属于慢作用、紧密结合的抑制剂。此外,TAP在体外试管内也具有抗凝活性,能延长凝血酶原时间、白陶土部份凝血活酶时间和改良Stypven时间,并与其浓度相关。     

人凝血因子V的纯化

目的 分离纯化人血浆中凝血因子V(FV),为研制FV单克隆抗体提供抗原.方法 在1 L新鲜冰冻血浆中加入酶抑制剂Benzamidine hydrochloride、二异丙基氟磷酸(DFP)和胰蛋白酶抑制剂,使其终浓度分别达到2mmol/L、1 mmol/L和50 mg/L;采用柠檬酸钡吸附、聚乙二醇6000分级沉淀、DEAE-Sepharose FF离子交换层析和Sephacryl-S300凝胶过滤层析,对血浆中FV进行分离纯化;采用凝血测定一期法检测FV活性;采用紫外分光光度法测定纯化FV的蛋白含量;采用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察FV的纯化情况.结果 经过对血浆前处理和两步层析后,FV活性回收率为(11.21±3.54)%,比活性为(30.46±0.31)U/mg,浓缩倍数为(2 538.33±25.83)倍(n=3);SDS-PAGE图谱显示:原料血浆添加DFP的离子交换层析样品较之未加DFP的样品在330 kD处有更粗更清晰的蛋白条带,最终纯化的FV样品仅在330 kD附近有1条较深的蛋白条带.结论 本FV纯化方法能够较有效地浓缩血浆中的FV.     

人D-二聚体分离纯化及鉴定

目的 分离纯度较高和人D-二聚体(D-dimer),为制备抗人D-二聚体单克隆抗体提供可靠抗原.方法 以凝血酶、凝血因子Ⅻ等处理人纤维蛋白原,得到交联纤维蛋白,经纤溶酶消化,得到交联纤维蛋白降解产物(FDP);对FDP作Sephacryl S-300凝胶过滤层析和聚焦层析,分离纯化得到D-dimer.采用免疫比浊法测定D-dimer 含量、染料结合法测定纯化蛋白含量,以SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察D-dimer的纯化情况,Weston Blot法确认D-dimer的存在.结果 制备得到的FDP SDS-PAGE图谱显示有多条蛋白条带,1条分子量为180 ～250kD蛋白条带在Western Blot图谱中显示可以被D-dimer单克隆抗体特异性地识别.FDP经Sephacry S-300凝胶层析和聚焦层析后得到纯度较高的D-dimer(n=3):总蛋白含量比为(77.82±7.98)％,蛋白回收率(25.27±6.35)％;SDS-PAGE图谱显示:经过两步层析得到的终产物为分子量180～250 kD的蛋白;Westem Blot证实:经过Sephacryl S-300凝胶层析和聚焦层析处理,最终得到分子量为180～250 kD的终产物可被D-dimer单克隆抗体特异性识别.结论 采用Sephacryl S-300凝胶层析和聚焦层析能够分离纯化获得较高纯度的D-dimer,可作为制备D-dimer单克隆抗体的抗原.     

人胰型及唾液型淀粉酶的分离纯化与鉴定

目的 为获得高比活性、高纯度的人胰型及唾液型淀粉酶。方法 用硫酸铵分级沉淀、Ｓｅ ｐｈａｄｅｘ Ｇ－５０凝胶层析及ＤＥＡＥ－纤维素离子交换层析法分别从人胰腺及唾液中分离纯化人胰型及唾液型淀粉酶,并用聚丙烯酰胺凝胶电泳法进行纯度、酶活性及分子量鉴定。结果 获得了高比活性及高纯度的人胰型及唾液型淀粉酶,胰型及唾液型淀粉酶的表现分子量分别为５５ＫＤ和５７ＫＤ。结论 本方法是获得高纯度及高比活性人胰型及唾     

“乐久精”中神经生长因子分子量的研究

“乐久精”是治疗精神分裂症阴性症状的新药，它是由白眉蝮蛇乌苏里亚种的蛇毒中分离纯化而制得。本研究用ＳＤＳ－凝胶丙烯酰胺电泳测定了该药的成分及其分子量，结果表明该分中含有神经生长因子，类凝血酶和舒缓激肽，其分子量分别为４３０００，３３５００和２１０００。     

C1酯酶抑制剂分离纯化工艺研究

目的 研究以人凝血酶原复合物(PCC)制备过程废弃洗涤液为原料分离纯化C1酯酶抑制剂(C1-esterase inhibitor, C1-INH)的工艺。方法 通过聚乙二醇(Polyethylene glycol, PEG)4000沉淀及阳离子层析两步法分离纯化C1-INH。调整原料pH及PEG4000浓度考察PEG4000沉淀法最佳条件;PEG沉淀后离心,上清液处理后作为阳离子交换层析的上样液,使用Fractogel EMD SE HiCap(M)凝胶及CM Sepharose FF凝胶进行离子交换层析,通过比较C1-INH活性收率及比活性选择最适合的凝胶及分离条件。结果 在pH6.1的条件下,PEG4000质量分数14%时,搅拌10min后离心,C1酯酶抑制剂回收率≈70%,铜蓝蛋白(ceruloplasmin, CER)去除率>95%;使用Fractogel EMD SE HiCap(M)凝胶进行阳离子交换层析,洗脱液盐浓度为0.25M氯化钠时,C1酯酶抑制剂活性收率>80%,比活性>5IU/mg。结论 以PCC制备过程中废弃洗涤液为原料,经过PEG沉淀和Fra...     

狼疮抗凝物抑制蛋白C途径引起血栓的机制研究

目的 从狼疮抗凝物（LA）抑制蛋白C（PC）途径研究系统性红斑狼疮（SLE）患者发生血栓的机制。方法 依整磷脂酰乙醇胺（PE）活化的蛋白C（APC）活性抑制试验（改良的dRVVT方法）,将正常人与SLE患者IgG预先与PE孵育后再加入纯化的试验体系中,观察是否抑制APC活性。结果 试验体系中APC的抗凝活性由于加入PE而增强。LA可抑制依赖PE的APC抗凝活性;且与其浓度成正比。SLE伴LA阳性的血栓患者的LA－IgG对APC抑制程度比LA阴性及无血栓的SLE患者更明显;而正常人IgG对APC无抑制作用。结论 LA通过干扰PE抑制PC途径可能SLE患者发生血栓的重要原因。     

Amidolytic kinetic assay of protein C by selective spectrophotometry in a centrifugal analyzer

This rapid, simple amidolytic assay of protein C activity in whole plasma involves activation by protein C activator from the venom of Agkistrodon contortrix contortrix (Protac) and use of a Cobas Fara spectrophotometer programmed for kinetic assay. Plasma is incubated with activator venom in the presence or absence of antibody to human protein C in the instrument, chromogenic substrate (S-2366) is added, and the absorbance is measured at 405 nm. The difference between the absorbance of the sample plasma with and without antibody to human protein C correlated well with protein C antigen as assayed by enzyme-linked immunosorbent assay (ELISA) and the Laurell rocket technique in normal subjects, patients being treated with warfarin, and patients with liver cirrhosis or disseminated intravascular coagulation. Our mean value for protein C in normal subjects is 115.9 (SD 16.7)% for amidolytic activity, 103.0 (SD 17.4)% for ELISA, and 97.2 (SD 18.1)% for the rocket technique. The high value for normal subjects presumably includes some nonspecific amidolytic activity activated by the activator venom, as indicated by measurable activity in immuno-depleted protein C-deficient plasma. Within-run and between-run CVs were less than 5% at low, normal, and high concentrations of protein C amidolytic activity.     

Formation and Excretion of Pyrrole-2-Carboxylate in Man

A detailed investigation of the purification of pyrrole-2-carboxylate (PCA) from human and rat urine indicates that previously reported mean values overestimate the correct quantity of free PCA by a factor of approximately three for rat urine and approximately five for human urine. Although several criteria of purity were satisfied by a previous method, pyrrole-reactive impurities were still present in the final fractions. These impurities are separated from PCA by chromatography through an amino acid analyzer ion-exchange resin. With the corrected method, normal human values for endogenous urinary PCA in 16 individuals averaged 0.51 mumol/day, with a range of 0.20-1.3 mumol and a SD of 0.31 mumol. The probable source of human PCA is free hydroxy-L-proline, as inferred from the high value for PCA in the urine of a subject with hereditary hydroxyprolinemia, and from the threeto eightfold elevation in PCA excretion by two normal subjects after a large oral load of hydroxyl-L-proline. Subcutaneous administration of [2-(14)C]PCA to a single human subject indicated almost complete conversion of the exogenous compound to derivatives, which are largely excreted in the urine. Data are discussed suggesting that much or all of the PCA in human urine may be formed in urine from a labile precursor, presumably Delta(1)-pyrroline-4-hydroxy-2-carboxylate.     

Radioimmunoassay of blood bradykinin: purification of blood extracts to prevent cross-reaction with endogenous kininogen

Alcoholic extracts of blood collected for measurement of circulating bradykinin were analysed for co-extracted kininogen which would artificially elevate measured bradykinin levels. The blood extract contained a glycoprotein with identical chromatographic properties to kininogen. Bradykinin immunoreactivity in the extract eluted as a single peak from an immunoaffinity column of anti-bradykinin IgG bound to Sepharose identically to both bradykinin and kininogen showing immunoidentity of these substances. Affinity chromatography on Concanavalin A-Sepharose or ion-exchange chromatography on CM-Sephadex C25 separated bradykinin and the glycoprotein which again eluted identically to kininogen. The decrease in measured values of blood bradykinin after purification of the extract on CM-Sephadex C25 was a similar amount to that calculated as cross-reactivity with the amount of co-extracted kininogen. Hence radioimmunoassay of bradykinin in ethanolic extracts of blood is inaccurate owing to the presence of co-extracted kininogen, and additional purification steps such as chromatography on CM-Sephadex C25 are mandatory for accurate assay by ligand binding techniques.     

C1r, subunit of the first complement component: purification, properties, and assay based on its linking role

A method to obtain C1r, a subunit of the first complement component, in a highly purified state has been described for the first time. The stepwise method starts with a neutral euglobulin precipitation, after diethylaminoethyl- and carboxymethyl-cellulose chromatography and a final preparative polyacrylamide electrophoresis step. Such C1r preparations are devoid of C1q and C1s activities and show only one protein band on analytic polyacrylamide electrophoresis. Rabbits injected with this preparation produced antisera showing only one precipitation band. The stability of C1r activity was determined under different conditions, and C1r was found to be labile at 37 degrees C, pH 7-8 and low ionic strength.The electrophoretic mobility of purified C1r is that of a beta-globulin on disc acrylamide electrophoresis and on agarose electrophoresis at pH 8.6. Its molecular weight as estimated by sephadex chromatography is 168,100.A sensitive hemolytic assay based on the property of C1r to link C1s to C1q and thereby to generate macromolecular C[unk]1 is described. The number of C[unk]1 molecules generated is stoichiometrically related to the concentration of C1r for a fixed C1q and C1s concentration provided that the titration is carried out below the plateau zone. Macromolecular C1 can be separated from free C1s as the former is cell bound.This method of purification and assay should allow the development of monospecific antisera and further chemical study of C1r.     

中药蛇伤散联合温针灸治疗蝮蛇咬伤肢体肿胀的临床观察

目的观察中药蛇伤散联合温针灸对蝮蛇咬伤后肢体软组织肿胀消退的疗效,及对横纹肌损害相关因子的影响。方法蝮蛇咬伤致肢体肿胀患者120例,随机分为观察组与对照组,每组各60例。对照组给予蝮蛇咬伤常规治疗,观察组在常规治疗基础上联合中医外治(中药蛇伤散外涂、温针灸)。观察比较两组患者治疗后伤侧与健侧肢体的周径差、软组织肿胀消退时间及肢体功能恢复情况、横纹肌损害相关因子(hs-CRP、CK、Mb)水平的变化。结果治疗后第1、2天,两组伤肢与健肢的周径差比较,差异无统计学意义(P>0.05)。治疗后第3、4、5天,观察组伤肢与健肢的周径差明显低于对照组,伤肢肿胀消退时间及疗程与对照组相比明显缩短,差异均有统计学意义(P<0.05)。观察组横纹肌损害相关因子恢复优于对照组,差异有统计学意义(P<0.05)。结论中药蛇伤散联合温针负可促进蝮蛇咬伤后软组织肿胀的消退,加快横纹肌损害相关因子水平恢复,促进伤肢功能恢复,缩短疗程。     

抗磷脂综合征合并静脉血栓和蛋白C活性异常1例研究报告

目的：探讨抗磷脂综合征（antiphospholipid syndrome，APS）患者静脉血栓形成的原因。方法：对1例APS患者用发色底物法测定蛋白C活性（PC：A）、蛋白S活性（PS：A）和抗凝血酶活性；用ELISA方法测定PC、血浆纤溶酶原、血浆组织型纤溶酶原激活物、血浆纤溶酶原激活抑制物-1、α2-抗纤溶酶抗原和抗心磷脂抗体（ACA）。活化蛋白C抵抗（APC—R）检测结果以受检者血浆加入APC后的APTT与未加APC血浆的APTT的比值表示，比值〈2．0时为APC-R阳性。狼疮抗凝物质（LA）检测使用以dRVVT为基础的商品化试剂盒。采用PCR扩增和直接测序，检测PC的基因及FVLeiden和凝血酶原G20210A的突变。结果：本例患者LA和APC—R阳性，PC：A降低，PC抗原量增加，其他结果正常．PC基因所有外显子测字结果正常，FVLeiden突变和凝血酶原G20210A突变未检出。结论：LA可能通过抑制PC途径导致患者发生血栓，联合检测ACA、APC-R、抗凝蛋白抗原及活性有利于血栓性疾病的病因学诊断。     

Fast functional assay of protein C in whole plasma using a snake venom activator: evaluation in patients with congenital and acquired protein C deficiencies

Both anticoagulant and amidolytic activities of protein C (PC) were measured using a snake venom activator in 4 patients with hereditary PC deficiency, 37 with disseminated intravascular coagulation (DIC), and 30 under stabilized warfarin therapy. The results were compared with those obtained by an immunological assay (ELISA). PC levels measured by different functional and immunological assays were very close in patients with hereditary PC deficiency and DIC. In patients under stable oral anticoagulant therapy, there was no detectable difference between amidolytic activity and antigen levels of PC in each patient plasma, whereas a decrease in anticoagulant activity was much more pronounced. These results indicate that the present activity assays measure PC specifically, and that the snake venom activator is capable of activating both carboxylated and hypocarboxylated forms of PC, but only anticoagulant assay can evaluate the physiological PC function in vitamin K-deficient states.     

重组人凝血因子Ⅶ的纯化与鉴定

目的建立重组人凝血因子Ⅶ(rhFⅦ)的纯化方法。方法首先将制备并纯化的单克隆抗体与CNBr-Sepharose 6B Fast Flow介质偶联,制备单克隆抗体亲和层析柱,并用rhFⅦ标准品验证层析柱的层析效果;采用DE-AE-Sephadex A-50吸附细胞表达的上清,对吸附产物用Sephadex G-25脱盐柱做脱盐处理;然后用单克隆抗体亲和层析柱做亲和层析;通过SDS-PAGE、Western Blot等试验测定纯化产物的纯度;通过凝血酶原时间(PT)测定纯化产物的促凝活性。结果制备出rhFⅦ单克隆抗体亲和层析介质;细胞表达产物经DEAE-Sephadex A-50、Sephadex G-25脱盐和亲和层析纯化后,经SDS-PAGE鉴定获得了分子量为50kD的单一蛋白洗脱峰;纯化所获得的重组蛋白具有促凝活性,促凝时间(DT)为52s。结论建立了纯化rhⅦ的亲和层析方法 ,为rhFⅦ的研制奠定了基础。